Effect of the NF-κB/iNOS Signaling Pathway on Spiral Ganglion in Mouse Model of Sensorineural Hearing Loss
-
摘要:目的 探究在3-硝基丙酸(3-NP)诱导的感音神经性听力损失小鼠模型中核转录因子κB(NF-κB)/诱导型一氧化氮合酶(iNOS)信号通路的表达水平变化对听力损失的影响。方法 本研究采用鼓室注射法进行动物建模。将C57BL/6雄性小鼠分为3个组,每组9只。3-NP组(鼓室内注射3-NP溶液),3-NP+EVP4593组(鼓室内注射3-NP溶液,腹腔注射NF-κB抑制剂EVP4593溶液),以及对照组〔鼓室内注射磷酸缓冲液(PBS)〕。在注射前后进行听性脑干反应检测。4周后取出耳蜗,并进行NF-κB p65、RelB、iNOS、Caspase-3抗体的免疫组织化学及实时荧光染色定量(qRT)-PCR检测。结果 3-NP组小鼠反应阈值较对照组和3-NP+EVP4593组高(P<0.05),且3-NP+EVP4593组的听阈比对照组高(P<0.05)。免疫荧光染色和qRT-PCR结果显示3-NP暴露可导致螺旋神经节区域NF-κB p65和RelB、iNOS表达较对照组增加(P<0.05),其表达可随着EVP4593的应用而减少(P<0.05)。3-NP组螺旋神经节细胞Caspase-3表达高于对照组,3-NP+EVP4593组Caspase-3的表达较3-NP组减少(P<0.05)。结论 3-NP可通过激活NF-κB/iNOS信号通路导致感音神经性听力损失模型小鼠的耳蜗螺旋神经节发生炎症反应,这可能在感音神经性听力损失的发病中起重要作用。Abstract:Objective To explore the effect of changes in the expression level of necorsis factor (NF)-κB/inducible nitric oxide synthase (iNOS) signaling pathway on hearing loss in a mouse model of sensorineural hearing loss (SNHL) induced by 3-nitropropionic acid (3-NP).Methods The animal model was established by tympanic injection. C57BL/6 male mice were divided into three groups, 3-NP group receiving tympanic injection of 3-NP solution, 3-NP+EVP4593 group receiving tympanic injection of 3-NP solution and intraperitoneal injection of EVP4593 solution, and a control group receiving tympanic injection of phosphate buffered saline (PBS). Auditory brainstem response (ABR) was tested before and after injection. After 4 weeks, the cochlea was harvested and immunohistochemistry and qRT-PCR of NF-κB p65, RelB, iNOS, and Caspase-3 were conducted accordingly.Results The hearing thresholds of the 3-NP group were higher than those of the control group and the 3-NP+EVP4593 group (P<0.05), and the hearing thresholds of the 3-NP+EVP4593 group were also higher than those of the control group (P<0.05). Immunofluorescence staining and qRT-PCR results showed that 3-NP exposure caused an increase in the expressions of NF-κB p65, RelB, and iNOS in the spiral ganglion in comparison with those of the control group (P<0.05), and their expressions decreased with the administration of EVP4593 (P<0.05). The expression of Caspase-3 in the spiral ganglion cells in the 3-NP group was higher than that in the control group, while in the 3-NP+EVP4593 group, it was lower than that in the 3-NP group (P<0.05).Conclusion This study found that, by activating the NF-κB/iNOS signaling pathway, 3-NP may cause inflammation in the spiral ganglion of the cochlear in the SNHL model mice, which may play an important role in the pathogenesis of SNHL.
-
Keywords:
- 3-nitropropionic acid /
- Sensorineural hearing loss /
- Spiral ganglion /
- NF-κB /
- iNOS
-
-
表 1 qRT-PCR引物序列
Table 1 Primer sequences for qRT-PCR
Target gene Forward primer (5′ to 3′) Reverse primer (5′ to 3′) Production length/bp NF-κB p65 CTTCCTCAGCCATGGTACCTCT CAAGTCTTCATCAGCATCAAACTG 126 RelB CTTTGCCTATGATCCTTCTGC GAGTCCAGTGATAGGGGCTCT 122 iNOS CTATCAGGAAGAAATGCAGGAGAT GAGCACGCTGAGTACCTCATT 125 GAPDH GGTGAAGGTCGGTGTGAACG CTCGCTCCTGGAAGAATGGTG 121 表 2 耳蜗螺旋神经节iNOS、NF-κB 65和RelB的表达平均荧光强度值及基因表达变化
Table 2 The mean fluorescence intensity and expression trend of iNOS, NF-κB p65, and RelB in the spiral ganglion
Index Group n Mean fluorescence intensity (protein) n Relative expression (gene) iNOS Control 6 62.04±1.81# 3 1.05±0.07# 3-NP 6 82.99±2.61 3 1.80±0.04 3-NP+EVP4593 6 58.87±1.98# 3 1.15±0.06# NF-κB p65 Control 6 70.03±2.94# 3 0.95±0.09# 3-NP 6 95.27±2.15 3 1.85±0.04 3-NP+EVP4593 6 68.21±3.52# 3 1.24±0.17#, Δ RelB Control 6 35.50±3.95# 3 1.10±0.03# 3-NP 6 104.40±4.66 3 2.20±0.10 3-NP+EVP4593 6 66.00±3.88#, Δ 3 1.20±0.16# # P<0.05, vs. 3-NP group; Δ P<0.05, vs. control group. -
[1] YAMAMOTO H, OMELCHENKO I, SHI X, et al. The influence of NF-kappaB signal-transduction pathways on the murine inner ear by acoustic overstimulation. J Neurosci Res,2009,87(8): 1832–1840. DOI: 10.1002/jnr.22018
[2] DOHI T, KAWASHIMA R, KAWAMURA Y I, et al. Pathological activation of canonical nuclear-factor κB by synergy of tumor necrosis factor α and TNF-like weak inducer of apoptosis in mouse acute colitis. Cytokine,2014,69(1): 14–21. DOI: 10.1016/j.cyto.2014.05.001
[3] CONTI A, MISCUSI M, CARDALI S, et al. Nitric oxide in the injured spinal cord: Synthases cross-talk, oxidative stress and inflammation. Brain Res Rev,2007,54(1): 205–218. DOI: 10.1016/j.brainresrev.2007.01.013
[4] WATANABE K, INAI S, JINNOUCHI K, et al. Nuclear-factor kappa B (NF-kappa B)-inducible nitric oxide synthase (iNOS/NOS Ⅱ) pathway damages the stria vascularis in cisplatin-treated mice. Anticancer Res,2002,22(6C): 4081–4085.
[5] MASUDA M, NAGASHIMA R, KANZAKI S, et al. Nuclear factor-kappa B nuclear translocation in the cochlea of mice following acoustic overstimulation. Brain Res,2006,1068(1): 237–247. DOI: 10.1016/j.brainres.2005.11.020
[6] SONE M, HAYASHI H, YAMAMOTO H, et al. Upregulation of HSP by geranylgeranylacetone protects the cochlear lateral wall from endotoxin-induced inflammation. Hear Res,2005,204(1-2): 140–146. DOI: 10.1016/j.heares.2005.01.012
[7] YIN H Y, MA X F, LIU F, et al. Protective effect of geranylgeranylacetone on cisplatin ototoxicity. Chemotherapy,2009,55(1): 1–5. DOI: 10.1159/000166382
[8] CHANG M Y, RHEE J, KIM S H, et al. The protective effect of Egb 761 against 3-nitropropionic acid-induced hearing loss: The role of Sirtuin 1. Clin Exp Otorhinolaryngol,2018,11(1): 9–16. DOI: 10.21053/ceo.2017.00626
[9] HOYA N, OKAMOTO Y, KAMIYA K, et al. A novel animal model of acute cochlear mitochondrial dysfunction. Neuroreport,2004,15(10): 1597–1600. DOI: 10.1097/01.wnr.0000133226.94662.80
[10] TIAN C, KIM Y H, KIM Y C, et al. Korean red ginseng ameliorates acute 3-nitropropionic acid-induced cochlear damage in mice. Neurotoxicology,2013,34: 42–50. DOI: 10.1016/j.neuro.2012.10.008
[11] GERONDAKIS S, GRUMONT R, GUGASYAN R, et al. Unravelling the complexities of the NF-kappaB signalling pathway using mouse knockout and transgenic models. Oncogene,2006,25(51): 6781–6799. DOI: 10.1038/sj.onc.1209944
[12] VALLABHAPURAPU S, KARIN M. Regulation and function of NF-kappaB transcription factors in the immune system. Annu Rev Immunol,2009,27: 693–733. DOI: 10.1146/annurev.immunol.021908.132641
[13] HOFFMANN A, BALTIMORE D. Circuitry of nuclear factor kappaB signaling. Immunol Rev,2006,210: 171–186. DOI: 10.1111/j.0105-2896.2006.00375.x
[14] BAUD V, KARIN M. Is NF-kappaB a good target for cancer therapy? Hopes and pitfalls. Nat Rev Drug Discov,2009,8(1): 33–40. DOI: 10.1038/nrd2781
[15] KEARNS J D, HOFFMANN A. Integrating computational and biochemical studies to explore mechanisms in NF-kappaB signaling. J Biol Chem,2009,284(9): 5439–5443. DOI: 10.1074/jbc.R800008200
[16] LISAKOVSKA O, SHYMANSKYY I, MAZANOVA A, et al. Vitamin D3 protects against prednisolone-induced liver injury associated with the impairment of the hepatic NF-κB/iNOS/NO pathway. Biochem Cell Biol,2017,95(2): 213–222. DOI: 10.1139/bcb-2016-0070
-
期刊类型引用(10)
1. 陆杰华. 中国老龄健康研究的学科分野、核心议题及其展望. 中国社会科学评价. 2024(04): 73-81+156-157 . 百度学术
2. 梁琼,张晓波,宋雪茜. 中国老年人口健康水平空间分布及影响因素. 中国老年学杂志. 2022(08): 1994-2000 . 百度学术
3. 李英杰,戴文旭. 农村老年人自评健康的影响因素研究. 农村经济与科技. 2022(17): 205-208 . 百度学术
4. 赵艳芳,钱皎,郭威,钱治军,涂博祥,王睿,吴骋. 某部基层战士自评健康状况调查. 武警医学. 2021(04): 296-299 . 百度学术
5. 李桐瑶,邴婧莹,李熹,薛乔丹,张悦,李润楠,王颖,孙宏. 我国独居老人自评健康状况及其影响因素研究. 现代预防医学. 2021(11): 2027-2031 . 百度学术
6. 张检,蔡金龙,何中臣,周学馨,唐贵忠. 重庆市农村留守老人自评健康状况及影响因素分析. 现代预防医学. 2020(12): 2215-2218+2241 . 百度学术
7. 杨博,张楠. 流动老年人健康自评的性别差异:基于健康双因素的多层模型研究. 人口与发展. 2019(02): 20-30 . 百度学术
8. 周超,徐永利,王依明. 公共服务水平对我国老年人健康状况的影响方式及其地区差异——基于CGSS2015调查数据的实证分析. 江苏建筑. 2019(S1): 127-132 . 百度学术
9. 王玲,杨洋,曾宪堡,唐贵忠,唐晓君,何中臣. 重庆市农村老年人自评健康影响因素分析. 现代预防医学. 2018(15): 2794-2798 . 百度学术
10. 张文娟,王东京. 中国老年人口的健康状况及变化趋势. 人口与经济. 2018(04): 86-98 . 百度学术
其他类型引用(12)